Respiratory syncytial virus (RSV) is a Parmixovirus of the Pneumovirus genus which commonly infects the upper and lower respiratory tract. It is so contagious that by age two, a large percentage of children have been infected by it. Moreover, by age four, virtually all humans have an immunity to RSV.
Typically, RSV infections are mild, remaining localized in the upper respiratory tract and causing symptoms similar to a common cold which require no extensive treatment. However, in some subjects, e.g., immunosuppressed individuals such as infants, elderly persons or patients with underlying cardiopulmonary diseases, the virus may penetrate to the lower respiratory tract requiring hospitalization and breathing support. In some of these cases, RSV infection may cause permanent lung damage or even be life threatening. In the United States alone, RSV results in about 90,000 hospitalizations each year, and results in about 4500 deaths.
RSV appears in two major strain subgroups, A and B, primarily based on serological differences associated with the attachment glycoprotein, G. The major surface glycoprotein, i.e., the 90 kD G protein, can differ up to 50% at the amino acid level between isolates Johnson et al, Proc. Natl. Acad. Sci. (1987), 84, 5625-5629. By contrast, a potential therapeutic target, the 70 kD fusion (F) protein, is highly conserved across different RSV strains, about i.e., 89% on the amino acid level Johnson et al. J. Gen. Virol.(1988), 69, 2623-2628, Johnson et al. J. Virol. (1987), 10, 3163-3166, P. L. Collins. Plenum Press. NY (1991), 103-162. Moreover, it is known that antibodies elicited against F-protein of a given type are cross-reactive with the other type.
The F-protein is a heterodimer, generated from a linear precursor, consisting of disulfide-linked fragments of 48 and 23 kD respectively Walsh et al, J. Gen. Virol, (1985), 66, 401-415. Inhibition of syncytia formation by polyclonal antibodies is associated with significant reaction to the 23 kD fragment.
As noted, while RSV infections are usually mild, in some individuals RSV infections may be life threatening. Currently, severe RSV infection is treated by administration of the antiviral agent Ribavarin. However, while Ribavarin exhibits some efficacy in controlling RSV infection, its use is disfavored for several reasons. For example, it is highly expensive and may be administered only in hospitals. Other known RSV treatments only treat the symptoms of RSV infection and include the use of aerosolized bronchodilators in patients with bronchiolitis and corticosteroid therapy in patients with bronchiolitis and RSV pneumonia.
To date, RSV vaccines intended to boost antiviral protective antibodies have been largely unsuccessful. For example, a vaccine based on formalin-inactivated RSV that was tested approximately 25 years ago, induced antibodies that were deficient in fusion inhibiting activity Murphy et al. Clinical Microbiology (1988), 26, 1595-1597, and sometimes even exacerbated the disease. This may potentially be explained to the inability of the formalin inactivated virus to induce protective antibodies. While high antibody titers were measured in vaccine recipients, specific protective titers were lower than in the control population. This may be because formalin inactivated RSV does not display the necessary conformational epitopes required to elicit protective antibodies.
While there is no known effective RSV vaccine to date, there exists some clinical evidence that antibody therapy may confer protection against RSV infection in susceptible individuals, and may even clear an existing RSV infection. For example, it has been reported that newborn infants show a low incidence of severe bronchiolitis, which is hypothesized to be attributable to the presence of protective maternal antibodies Ogilvie et al. J. Med Virol (1981), 7, 263-271. Also, children who are immune to reinfection exhibit statistically higher anti-F-protein titers than those who are reinfected. Moreover, intravenous immune globulin (IVIG) prepared from high titer RSV-immune donors reduces nasal RSV shedding and improves oxygenation Hemming et al. Anti. Viral Agents and Chemotherapy (1987), 31, 1882-1886. Also, recent studies have suggested that the virus can be fought and lung damage prevented by administering RSV-enriched immune globulin (RSVIG) Groothuis et al. The New England J. Med. (1993), 329, 1524-1530, K. McIntosh. The New England J. Med. (1993), 329, 1572-1573, J. R. Groothuis, Antiviral Research, (1994), 23, 1-10, Siber et al. J. Infectious Diseases (1994), 169, 1368-1373, Siber et al. J. Infectious Diseases (1992), 165:456-463.
Similarly, some animal studies suggest that antibody therapy with virus neutralizing antibodies may confer protection against RSV or even clear an existing RSV infection. For example, in vitro neutralizing mouse monoclonal antibodies have been reported to protect mice against infection and also to clear established RSV infections Taylor et al. J. Immunology, (1984), 52, 137-142, Stott et al. "Immune Responses, Virus Infections and Disease, I.R.L. Press, London (1989), 85-104. Also, monoclonal antibodies to the F-protein of RSV have shown high efficacy in both in vitro and in vivo RSV models Tempest et al. Bio/Technology, (1991), 9, 266-271, Crowe et al. Proc. Natl. Acad. Sci. (1994), 91, 1386-1390, Walsh et al. Infection and Immunity, (1984), 43, 756-758, Barbas III, et al, Proc. Natl. Acad. Sci. (1992), 89, 10164-10168, Walsh. et al. J. Gen. Virol. (1986), 67, 505-513. Antibody concentrations as low as 520-2000 .mu.g/kg body weight have been reported to result in almost instant recovery in animal studies Crowe et al. Proc. Natl. Acad. Sci. (1994), 91, 1386-1390. Moreover, these monoclonal antibodies have been disclosed to neutralize both A and B strains, including laboratory strains and wildtype strains. These antibodies were administered either by injection Groothuis et al. The New England J. Med. (1993), 329, 1524-1530, Siber et al. J. Infectious Diseases (1994), 169, 1368-1373 or by aerosol Crowe et al. Proc. Natl. Acad. Sci. (1994), 91, 1386-1390.
Two different types of potentially therapeutic monoclonal antibodies to the RSV F-protein have been previously described in the literature, humanized murine antibodies Tempest et al, Biol. Technology, (1991) 9, 266-271, or true human antibodies (Fab fragments) Barbas III. et al, Proc. Natl. Acad. Sci. (1992), 89, 10164-10168. Humanized murine antibodies were generated by CDR grafting a cross-strain neutralizing murine anti-F-protein antibody onto a generic human Fc, as well as structural areas of the variable part. The human Fab fragments were produced by combinatorial library technology using human bone marrow cells obtained from an HV positive donor (immunocompromised). The therapeutic in vivo titers of the humanized and human RSV antibodies were 5 and 2 mg/kg body weight, respectively. It is noted, however, that the humanized antibodies were tested in a syncytia inhibition assay, whereas the human anti-RSV Fab fragments were assayed to determine their virus neutralization activity. Therefore, the results reported with the humanized and human anti-RSV antibodies are not directly comparable.
The Fab fragment generated by the combinatorial library technology were disclosed to be efficient in aerosol. This is probably because of the relatively small size of the molecule. These results are highly encouraging because a major target population for an RSV vaccine is infants. Therefore, aerosol is a particularly desirable mode of administration.
However, notwithstanding the previous published reports of humanized and Fab fragments specific to RSV, there still exists a significant need for improved anti-RSV antibodies having improved therapeutic potential, in particular anti-RSV antibodies which possess high affinity and specificity for the RSV F-protein which effectively neutralize and prevent RSV infection.
Antibody therapy can be subdivided into two principally different activities: (i) passive immunotherapy using intact non-labeled antibodies or labeled antibodies and (ii) active immunotherapy using anti-idiotypes for re-establishment of network balance in autoimmunity.
In passive immunotherapy, naked antibodies are administered to neutralize an antigen or to direct effector functions to targeted membrane associated antigens. Neutralization would be of a lymphokine, a hormone, or an anaphylatoxin, i.e., C5a. Effector functions include complement fixation, macrophage activation and recruitment, and antibody dependent cell mediated cytotoxicity (ADCC). Naked antibodies have been used to treat leukemia Ritz et al. S. F. Blood. (1981), 58, 141-152 and antibodies to GD2 have been used in treatments of neuroblastomas Schulz et al. Cancer Res. (1984), 44:5914 and melanomas Irie et al. Proc. Natl. Acad. Sci., (1986, 83:8694. Also, intravenous immune gamma globulin (IVIG) antibodies with high anti-RSV titers recently were used in experimental trials to treat respiratory distress caused by RSV infection Hemming et al. Anti. Viral Agents and Chemotherapy, (1987), 31, 1882-1886, Groothuis et al. The New England J. Med. (1993), 329, 1524-1530, K. McIntosh. The New England J. Med. (1993), 329, 1572-1573, J. R. Groothuis. Antiviral Research, (1994), 23, 1-10, Siber et al. J. Infectious Diseases (1994), 169, 1368-1373.
The therapeutic efficacy of a monoclonal antibody depends on factors including, e.g., the amount, reactivity, specificity and class of the antibody bound to the antigen. Also, the in vivo half-life of the antibody is a significant therapeutic factor.
Still another factor which may significantly affect the therapeutic potential of antibodies is their species of origin. Currently, monoclonal antibodies used for immunotherapy are almost exclusively of rodent origin Schulz et al. Cancer Res. (1984), 44:5914, Miller et al. Blood (1981), 58, 78-86, Lanzavecchia et al. J. Edp. Med. (1988), 167, 345-352, Sikora et al. Br. Med. Bull. (1984), 40:240, Tsujisaki et al. Cancer Research (1991), 51:2599, largely because the generation of rodent monoclonal antibodies uses well characterized and highly efficient techniques Kohler et al. Nature, (1975), 256:495, Galfre et al. Nature, (1977), 266:550. However, while rodent monoclonal antibodies possess therapeutic efficacy, they can present restrictions and disadvantages relative to human antibodies. For example, they often induce sub-optimal stimulation of host effector functions (CDCC, ADCC, etc.). Also, murine antibodies may induce human anti-murine antibody (HAMA) responses Schroff et al. Can. Res. (1985, 45:879-885, Shawler et al. J. Immunol. (1985), 135:1530-1535. This may result in shortened antibody half-life Dillman et al. Mod. (1986), 5, 73-84, Miller et al. Blood, (1983), 62:988-995 and in some instances may cause toxic side effects such as serum sickness and anaphylaxis.
In some subjects, e.g., heavily immunosuppressed subjects (e.g., patients subjected to heavy chemical or radiation mediated cancer therapy Irie et al. Proc. Natl. Acad. Sci. (1986), 83:8694, Dillman et al. Mod. (1986), 5, 73-84, Koprowski et al. Proc. Natl. Acad. Sci. (1984), 81:216-219), use of murine monoclonal antibodies causes limited negative side effects. By contrast, in patients with normal or hyperactive immune systems, murine antibodies, at least for some disease conditions may exhibit limited efficacy.
In an effort to obviate limitations of murine monoclonal antibodies, recombinant DNA techniques have been applied to produce chimeric antibodies Morrison et al. Proc. Natl. Acad. Sci. (1984), 81:216-219, Boulianne et al. Nature, (1984), 312, 644-646, humanized antibodies by "CDR grafting" Riechmann et al. Nature (1984), 332, 323-327 and "veneered" antibodies by substitution of specific surface residues with other amino acids to alleviate or eliminate antigenicity.
However, although such antibodies have been used successfully clinically Gillis et al. J. Immunol. Meth (1989), 25:191, they have proven cumbersome to produce. This is because the understanding of the requirements for optimal antigen recognition and affinity is not yet fully understood. Also, the human framework and the mouse CDR regions often interact sterically with a negative effect on antibody activity. Moreover, such antibodies sometimes still induce strong HAMA responses in patients.
Human antibodies present major advantages over their murine counterparts; they induce optional effector functions, they do not induce HAMA responses and host antigen-specific antibodies may lead to identification of epitopes of therapeutic value that may be too subtle to be recognized by a xenogeneic immune system Lennox et al. "Monoclonal Antibodies in Clinical Medicine." London: Academic Press (1982).
While human antibodies are highly desirable, their production is complicated by various factors including ethical considerations, and the fact that conventional methods for producing human antibodies are often inefficient. For example, human subjects cannot generally be adequately immunized with most antigens because of ethical and safety considerations. Consequently, reports of isolation of human monoclonal antibodies with useful affinities, .gtoreq.10.sup.8 molar to specific antigens are few McCabe et al. Cancer Research, (1988), 48, 4348-4353. Also, isolation of anti-viral human monoclonal antibodies from donor primed cells has proved to be unwieldy. For example, Gorny reported that only 7 of 14,329 EBV transformed cultures of peripheral blood mononuclear cells (PMBC's) from HIV positive donors resulted in stable, specific anti-HIV antibody producing cell lines Gorny et al. Proc. Natl. Acad. Sci. (1989), 86:1624-1628.
To date, most human anti-tumor antibodies have been generated from peripheral blood lymphocytes (PBLs) Irie et al. Br. J. Cancer, (1981), 44:262 or tumor draining lymph node lymphocytes Schlom et al. Proc. Natl. Acad. Sci. (1980), 77:6841-6845, Cote et al. Proc. Natl. Acad. Sci. (1983), 80:2026-2030 from cancer patients. However, such antibodies often react with intracellular, and thus therapeutically useless antigens Ho et al. In Hybridoma Technology, Amsterdam (1988), 37-57 or are of the IgM class McCabe et al. Cancer Research (1988), 48, 4348-4353, a class of antibodies with lesser ability to penetrate solid tumors than IgGs. Few of these human antibodies have moved to clinical trials Drobyski et al. R.C. Transplantation (1991), 51, 1190-1196, suggesting that the rescued antibodies may possess sub-optimal qualities. Moreover, since these approaches exploit the testing donor primed B cells, it is clear that these cells are not an optimal source for rescue of useful monoclonal antibodies.
Recently, generation of human antibodies from primed donors has been improved by stimulation with CD40 resulting in expansion of human B cells Banchereau et al. F. Science (1991), 251:70, Zhang et al. J. Immunol. (1990), 144, 2955-2960, Tohma et al. J. Immunol. (1991), 146:2544-2552 or by an extra in vitro booster step primer to immortalization Chaudhuri et al. Cancer Supplement (1994), 73, 1098-1104. This principle has been exploited to generate human monoclonal antibodies to Cytomegalovirus, Epstein-Barr Virus (EBV) and Hemophilus influenza with cells from primed donors Steenbakkers et al., Hum. Antibod. Hybridomas (1993), 4:166-173, Ferraro et al., Hum. Antibod. Hybridomas (1993), 4:80-85, Kwekkeboom et al., Immunological Methods (1993), 160 :117-127, with a significantly higher yield than obtained with other methods Gorny et al., Proc. Natl. Acad. Sci. (1989), 86:1624-1628.
Moreover, to address the limitations of donor priming, immunization and cultivation ex vivo of lymphocytes from healthy donors has been reported. Some success in generating human monoclonal antibodies using ex homine boosting of PBL cells from primed donors has been reported Maeda et al. Hybridoma (1986), 5:33-41, Kozbor et al. J. Immunol. (1984), 14:23, Duchosal et al. Nature (1992, 355:258-262. The feasibility of immunizing in vitro was first demonstrated in 1967 by Mishell and Dutton Mishell et al. J. Exp. Med (1967), 126:423-442 using murine lymphocytes. In 1973, Hoffman successfully immunized human lymphocytes Hoffman et al. Nature (1973), 243:408-410. Also, successful primary immunizations have been reported with lymphocytes from peripheral blood Luzzati et al. J. Exp. Med. (1975), 144:573:585, Misiti et al. J. Exp. Med. (1981), 154:1069-1084, Komatsu et al. Int. Archs. Allergy Appl. Immunol. (1986), 80:431-434, Ohlin et al. C.A.K. Immunology (1989), 68:325 (1989) tonsils Strike et al. J. Immunol. (1978), 132:1789-1803 and spleens, the latter obtained from trauma Ho et al. In Hybridoma Technology, Amsterdam (1988), 37-57, Boerner et al. J. Immunol. (1991), 147:86-95, Ho et al. J. Immunol. (1985), 135:3831-3838, Wasserman et al. J. Immunol. Meth. (1986), 93:275-283, Wasserman et al. J. Immunol. Meth. (1986), 93:275-283, Brams et al. Hum. Antibod. Hybridomas (1993), 4, 47-56, Brams et al. Hum. Antibod. Hybridomas (1993), 4, 57-65 and idiopathic thrombocytopenia purpura (ITP) patients Boerner et al. J. Immunol. (1991), 147:86-95, Brams et al. Hum. Antibod. Hybridomas (1993) 4, 47-56, Brams et al. Hum. Antibod. Hybridomas (1993), 4, 57-65, McRoberts et al. "In Vitro Immunization in Hybridoma Technology". Elsevier, Amsterdam (1988), 267-275, Lu et al. P. Hybridoma (1993), 12, 381-389.
In vitro immunization offers considerable advantages, e.g., easily reproducible immunizations, lends itself easily to manipulation of antibody class by means of appropriate cultivation and manipulation techniques Chaudhuri et al. Cancer Supplement (1994), 73, 1098-1104. Also, there is evidence that the in vivo tolerance to self-antigens is not prevalent during IVI Boerner et al. J. Immunol. (1991), 147:86-95, Brams et al. J. Immunol. Methods (1987), 98:11. Therefore, this technique is potentially applicable for production of antibodies to self-antigens, e.g., tumor markers and receptors involved in autoimmunity.
Several groups have reported the generation of responses to a variety of antigens challenged only in vitro, e.g., tumor associated antigens (TAAs) Boerner et al. J. Immunol. (1991), 147:86-95, Borrebaeck et al. Proc. Natl. Acad. Sci. (1988), 85:3995. However, unfortunately, the resulting antibodies were typically of the IgM and not the IgG subclass McCabe et al. Cancer Research (1988), 48, 4348-4353, Koda et al. Hum. Antibod. Hybridomas, (1990), 1:15 and secondary (IgG) responses have only been reported with protocols using lymphocytes from immunized donors. Therefore, it would appear that these protocols only succeed in inducing a primary immune response but require donor immunized cells for generation of recall responses.
Also, research has been conducted to systematically analyze cultivation and immunization variables to develop a general protocol for effectively inducing human monoclonal antibodies in vitro Boerner. J. Immunol. (1991) 147:86-95, Brams et al. Hum. Antibod. Hybridomas (1993), 4, 47-56, Lu et al. Hybridoma (1993), 12, 381-389. This has resulted in the isolation of human monoclonal antibodies specific for ferritin Boerner et al. J. Immunol. (1991), 147:86-95, induced by IVI of naive human spleen cells. Also, this research has resulted in a protocol by which de novo secondary (IgG) responses may be induced entirely in vitro Brams et al. Hum. Antibod. Hybridomas (1993), 4, 57-65.
However, despite the great potential advantages of IVI, the efficiency of such methods are severely restricted because of the fact that immune cells grow in monolayers in culture vessels. By contrast, in vivo germinal centers possessing a three-dimensional structure are found in the spleen during the active phases of an immune response. These three-dimensional structures comprise activated T- and B-cells surrounded by antigen-presenting cells which are believed by the majority of immunologists to compare the site of antigen-specific activation of B-cells.
An alternative to the natural splenic environment is to "recreate" or mimic splenic conditions in an immunocompromised animal host, such as the "Severe Combined Immune Deficient" (SCID) mouse. Human lymphocytes are readily adopted by the SCID mouse (hu-SCID) and produce high levels of immunoglobulins Mosier et al. Nature (1988), 335:256, McCune et al. L. Science (1988), 241, 1632-1639. Moreover, if the donor used for reconstitution has been exposed to a particular antigen, a strong secondary response to the same antigen can be elicited in such mice. For example, Duchosal et al. Duchosal et al. Nature (1992), 355:258-262 reported that human peripheral blood B-cells from a donor vaccinated with tetanus toxoid 17 years prior could be restimulated in the SCID environment to produce high serum levels, i.e., around 10.sup.4. They further disclosed cloning and expression of the genes of two human anti-TT antibodies using the lambda and the M13 phage combinatorial library approach Huse et al. R. A. Science (1989), 246:1275 from the extracted human cells. The reported antigen affinities of the antibodies were in the 10.sup.8 -10.sup.9 /M range. However, this protocol required donor primed cells and the yield was very low, only 2 clones were obtained from a library of 370,000 clones.
Therefore, previously the hu-SPL-SCID mouse has only been utilized for producing human monoclonal antibodies to antigens wherein the donor has either been efficiently primed naturally or by vaccination Stahli et al. Methods in Enzymology (1983), 92, 26-36, which in most cases involves exposure to viral or bacterial antigens. Also, the reported serum titer levels using the hu-SCID animal model are significantly lower than what is typically achieved by immunization of normal mice.
Additionally, two protocols have been described by which induction of primary antibody responses can be followed by induction of secondary antibody responses in hu-SCID mice using naive human lymphocytes. However, use of both of these protocols are substantially restricted. In the first protocol, primary responses are induced in hu-SCID mice into which human fetal liver, thymus and lymph nodes have been surgically implanted. However, this method is severely restricted by the limited availability of fetal tissue, as well as the complicated surgical methodology of the protocol McCune et al. L. Science (1988), 241, 1632-1639. In the second protocol, lethally irradiated normal mice were reconstituted with T- and B-cell depicted human bone marrow and SCID mouse bone marrow cells Lubin et al. Science, (1991), 252:427. However, this method is disadvantageous because it requires a four month incubation period. Moreover, both protocols result in very low antibody titers, i.e., below 10.sup.4.
Also, Carlson et al. Carlsson et al. J. Immunol. (1992), 148:1065-1071 described in 1992 an approach using PBMCs from an antigen (tetanus toxoid) primed donor. The cells were first depleted of macrophages and NK cells before being subjected to a brief in vitro cultivation and priming period prior to transfer into a SCID mouse. The hu-SPL-SCID mouse was then boosted with antigen. This method was reported to result in average TT specific human IgG titers of .congruent.10.sup.4 in the hu-SPL-SCID serum, with up to 5.times.10.sup.5 reported.
Production of human monoclonal antibodies further typically requires the production of immortalized B-cells, in order to obtain cells which secrete a constant, ideally permanent supply of the desired human monoclonal antibodies. Immortalization of B-cells is generally effected by one of four approaches: (i) transformation with EBV, (ii) mouse-human heterofusion, (iii) EBV transformation followed by heterofusion, and (iv) combinatorial immunoglobulin gene library techniques.
EBV transformation has been used successfully in a number of reports, mainly for the generation of anti-HIV antibodies Gorny, et al. Proc. Natl. Acad. Sci. (1989), 86:1624-1628, Posner, et al. J. Immunol. (1991), 146:4325-32. The main advantage is that approximately one of every 200 B-cells becomes transformed. However, EBV transformed cells are typically unstable, produce low amounts of mainly IgM antibody, clone poorly and cease making antibody after several months of culturing. Heterofusion Carrol, et al. J. Immunol. Meth. (1986), 89:61-72 is typically favored for producing hybridomas which secrete high levels of IgG antibody. Hybridomas are also easy to clone by limiting dilution. However, a disadvantage is the poor yield, i.e., .ltoreq.1 hybridomas per 20,000 lymphocytes Boerner. et al. J. Immunol. (1991), 147:86-95, Ohlin. et al. C.A.K. Immunology (1989), 68:325, Xiu-mei et al. Hum. Antibod. Hybridomas (1990), 1:42, Borrebaeck C.A.K. Abstract at the "Second International Conference" on "Human Antibodies and Hybridomas." Apr. 26-28, 1992, Cambridge, England. Combining EBV transformation followed by heterofusion offers two advantages: (i) human B-cells fuse more readily to the fusion partner after EBV transformation, and (ii) result in more stable, higher, producing hybridomas Ohlin. et al. Immunology (1989), 68:325, Xiu-mei. et al. Hum. Antibod. Hybridomas (1990), 1:42, Borrebaeck C.A.K. Absract at the "Second International Conference" on "Human Antibodies and Hybridomas." Apr. 26-28, 1992, Cambridge, England. The advantage of the final technique, i.e., combinatorial immunoglobulin gene library technique is the fact that very large libraries can be screened by means of the M13 Fab expression technology Huse. et al. Science (1989), 246:1275, William Huse. Antibody Engineering: A Practical Guide. Borrebaeck C.A.K. ed. 5:103-120 and that the genes can easily be transferred to a production cell line. However, the yield is typically extremely low, on the order of 1 per 370,000 clones Duchosal. et al. Nature (1992), 355:258-262.
Thus, based on the foregoing, it is apparent that more efficient methods for producing human monoclonal antibodies, in particular antibodies specific to RSV, would be highly advantageous. Moreover, it is also apparent that human antibodies specific to the RSV F-protein having superior binding affinity, specificity and effector functions than those currently available would also be highly desirable.